Metal chelates and methods of using them for time-resolved fluorescence

ABSTRACT

β-diketone fluorescent tags are disclosed, particularly those enabling the use of excitation energy in the near visible or visible spectrum. In some cases, these tags allow the use of cost-effective excitation devices such as LED&#39;s. The compounds form fluorescent chelates (complexes) with lanthanide(III) rare earth metal ions (such as Eu3+). The fluorescent complex may be included in a latex microparticle, such as a styrene latex particle. Ideally, the complex has an absorption maximum λ equal to or greater than 360 nm, and the compound is characterized by a pKa &lt;9.0. Kits and methods for detecting target molecules (e.g. immunoassays) are also disclosed. Such methods and kits typically use a ligand for binding to the target molecule and a labeling agent attached to the ligand. The fluorescent complexes described above are at least part of the labeling agent. Apparatus for detecting fluorescence from a sample includes an irradiating energy source that produces irradiating energy λ equal to or greater than 360 nm; a detector positioned to detect fluorescence from the sample; and a sample holder for holding the sample in position to be irradiated by the energy source. A light-emitting diode is preferably used as the irradiating energy source.

TECHNICAL FIELD

This invention is in the general field of fluorescent complexes,particularly those having chelated metal ions; it is also in the fieldof synthesis and in the field of the use of chelating compounds andmetal-ion complexes, for example as tags to enable detection (e.g. byinstrument) in an assay.

BACKGROUND

It is often desirable to use fluorescence to detect the presence of acompound. For example, assays (such as immunoassays) can be read bydetecting fluorescent energy emitted by a fluorescent tag associatedwith the compound being detected. Fluorescence tags are relatively easyto use, and they avoid hazards and procedures associated withradioactive tags. Useful applications for fluorescent detection include,without limitation, cell imaging, flow cytometry, immunohistochemistry,and immunoassays. Conventional fluorescent dyes include fluorescein,rhodamine, Texas Red and others.

A frequent problem in fluorescence-based assays is interference due tobackground fluorescence in the sample or reagents used in the assay.Because this background fluorescence often has a relatively shortlifetime and low stokes shift, the use of fluorescent tags with largestokes shift or very long lifetimes (time-resolved fluorescence) allowsthe detection of smaller amounts of the tag in the presence of largeamounts of the background. One method of time-resolved fluorescenttagging involves the use of chelated (using organic chelators)lanthanide metals.1-6 Lanthanide chelation complexes available todayrequire excitation with ultraviolet light (e.g., often below 340nm11-15), requiring complex and relatively expensive light sources, suchas a nitrogen laser. Two commercial products based on lanthanide timeresolved fluorescence are Perkin Elmer's DELFIA® and LANCE™ products(Perkin Elmer Bioproducts, Boston Mass.). The DELFIA® Eu-labelingreagent consists ofN1-(p-isothiocyanatobenzyl)-diethylenetriamine-N1,N2,N3,N4-tetraaceticacid (DTTA) chelated with Eu3+. The DTTA group forms a stable complexwith europium, and the isocyanate group reacts with a free amino groupon the protein to form a stable, covalent thiourea bond. The high watersolubility and stability of the chelate, in addition to the mildcoupling conditions of the isothiocyanate reaction, make it possible tolabel antibodies with up to 10-20 Eu/IgG The LANCE™ product is also anisocyanate (ITC) based chelating product.

The literature reports other chelates that are excitable at longerwavelengths. In some cases, these chelates exhibit relatively poorquantum yields and/or inefficient energy transfer from the chelatingcompound to the metal ion. For example, Eu (III) Schiff base complexesexhibit relatively low quantum yields when the absorption maximum occursat longer wavelengths.16 For some chelates, fluorescence is essentiallylimited to organic solvents, making them unattractive or impractical forbiological applications. Martinus et al described a Eu chelate withMichler's ketone [4,4′-bis(N,N-dimethylamino)benzophenone] (“MK”) withabsorption maximum at 414 nm. Again, complex formation occurs innon-coordinating solvents, and water molecules may compete with MK forlanthanide coordination sites.17

Steemers et al. were able to make Europium and Terbium complexes with aseries of calix[4]arenes with excitations extended to at least 350 nm.The reported quantum yields are relatively low and energy transfer isrelatively inefficient. It is believed that a significant fraction ofthe excited species are trapped by molecular oxygen resulting inquenching without contributing to luminescence.18 Werts et al. disclosecomplexes of lanthanides with Fluorexon(4′,5′bis[N,Nbis(carboxymethyl)aminomethyl]fluorscein) which can beexcited with visible light.19 However these chelates reportedly haverelatively low quantum yields (between 1.7-8.9×10-4) due tonon-radiative deactivation.

The anion (compound 1a, below) of the aromatic 1,3-diketone,2-naphthoyltrifluoroacetone (NTA, compound 1, below) forms a highlyfluorescent Eu chelate in aqueous solvent, in the presence of thesynergistic agent, tri-n-octyl-phosphene oxide (TOPO).20,21

SUMMARY

The invention features fluorescent tags exhibiting a desirablecombination of one or more of the following characteristics: longlifetime, narrow band width, large stokes shift, high quantum yield, andavoidance of detrimental phenomena such as photobleaching orself-quenching. Preferably, the invention enables the use of excitationenergy in the near visible or visible spectrum, allowing the use ofcost-effective excitation devices such as light-emitting diodes or diodelasers, making the technology more practical for commercial use.

In general, the invention features a β-diketone compound having one ofthe formulas A, B or C as provided in claim 1, below. Preferably, thecompound forms fluorescent chelates with lanthanide (III) rare earthmetal ions (such as Eu3+), and, therefore, the invention also featurescomplexes of the compounds with such ions. The invention also featuresnon-fluorescent chelators, such as tri-n-octyl-phosphene oxide (TOPO) orNH₃, in a composition with the above fluorescent chelators. The rareearth metal ion may be included in such compositions or added to them ina later step.

In one embodiment, the fluorescent complex is incorporated on or into amicroparticle, such as a styrene latex particle or on a colloidal goldparticle. Preferably, the complex has an absorption maximum at λ equalto or greater than 360 nm, and the compound is characterized by a pKa<9.0 that allows the complex to be stable at physiological pH.

The complexes are particularly useful tags for detecting targetmolecules, and the invention features kits and methods for suchdetection. Such methods and kits typically use a ligand for binding tothe target molecule and a labeling agent attached to the ligand. Thefluorescent complexes described above are at least part of the labelingagent. The target molecule is reacted with the complex-labeled ligand,and the presence of the target is detected by irradiating with energy λequal to or greater than 360 nm, and then detecting emission from thefluorescent complex as an indication that the target molecule ispresent.

Apparatus for detecting fluorescence from a sample includes anirradiating energy source that produces irradiating energy λ equal to orgreater than 360 nm; a sample holder for holding the sample in positionto be irradiated by the energy source, a detector positioned to detectfluorescence from the sample; and detector circuitry to allowinstantaneous or time-resolved detection of the fluorescence. Theinvention may permit the use of a semiconductor light source such as alight-emitting diode or laser diode as the irradiating energy source.

Specifically preferred compounds according to the formula in claim 1 arethose in which:

-   -   Ar₁=a napthyl, anthrenyl, or phenanthrenyl group or a        substituted napthyl (for example a 2′ naphthyl), anthrenyl, or        phenanthrenyl group;    -   Ar₂, Ar₃=a substituted phenyl group, e.g., a phenyl group        substituted by one or more of: —F; —CF₃; and —N(CH₃)₂;        where substitutions include C₁-C₅ alkyl, C₁-C₅ alkenyl, C₁-C₅        alkynyl groups, or substituted C₁-C₅ alkyl, C₁-C₅ alkenyl, C₁-C₅        alkynyl groups;

The details of one or more embodiments of the invention are set forth inthe accompanying drawings and the description below. Other features,objects, and advantages of the invention will be apparent from thedescription and drawings, and from the claims.

DESCRIPTION OF DRAWINGS

Figure I is a schematic diagram of the radiative processes of an Eu³+chelation complex.

Figure II is an excitation and emission spectrum of NTA, PNPD and NNPD.

Figure III shows the structures of various derivatives of compound 2.

Figure IV diagrams the coordination of a metal (M+) with certaincompounds.

Figure V compares time resolved versus continuous fluorescence asdescribed below.

Figure VI depicts results from immunoassay for feline leukemia virus.

Figure VII is a synthetic scheme for carboxy functionalized derivativesof PNPD and their conjugation to proteins.

Figure VIII is an excitation and emission spectrum of Eu-PNPD chelatecoated colloidal gold.

DETAILED DESCRIPTION

Two compounds were designed in which the trifluoromethyl group in NTA(1) was replaced with a phenyl ring (phenyl-3-naphthyl-1,3-propanedione,compound 2 or “PNPD”) and a naphthyl ring(1,3-di-naphthyl-1,3propanedione, compound 3 or “NNPD”). The structuresof these compounds and their corresponding enolates are shown below:

Compounds 2 and 3 were synthesized starting from 1′-acetonaphthone andtheir structures were characterized as described below. Fluorescentchelates of europium (III) chloride were prepared in 0.1 M borate bufferpH 9.0 with each of 2, and 3, respectively in presence of TOPO, and theexcitation and emission spectra were recorded on Perkin ElmerLuminescence Spectrophotometer model LS 50 and compared with that of NTA(compound 1). Spectral results (Figure II) demonstrate that the peakexcitation wavelengths for compounds 2 and 3 are 390 nm and 400 nmrespectively; both are shifted significantly form the peak at 330 nm forcompound 1 and can be excited with a semiconductor light source. Theextinction and fluorescence intensity of all of the compounds aresimilar. These compounds have high quantum yields coupled with stabilityin oxygenated solvents, suggesting efficient energy transfer from ligandto metal.

Based on these findings, derivatives of compound 2 were prepared; theirstructures are depicted in Figure III and their synthesis andcharacterization are described below.

All the compounds that are depicted in Figure III can form fluorescentchelates with europium(III)chloride in presence of TOPO. Interestingly,no significant spectral shifts were observed for derivatives withdifferent ring substituents such as fluorine, cyano, methoxy, andcarboxymethyl compared to PNPD. However, significant changes in pKa ofthe carbonyl groups were observed. As mentioned earlier, it is necessaryto form the enolate of the dicarbonyl compund for complex formation withmetal to occur. In the case of unsubstituted compound 2, the pKa for theenolic carbon is ˜pH 9.0 and hence it is necessary to maintain that highpH to form the complex and maintain its stability. This limits theability to successfully load the chelate into latex particles asdescribed below. However, the pKa of all the fluorinated derivatives issignificantly lower allowing us to work with the chelates over a rangeof pH's. For example the pKa for compound 4 is ˜4.2 which allowed us touse this compound to form a stable chelate above pH 4.5. It is usually(by not universally) desirable to stay below pH 9.0 to allow chelatestability under physiological assay conditions.

The excitation wavelength of the fluorescent chelate ofN,N-dimethylamino derivative of the PNPD (compound # 14 in Figure III)when loaded into latex particles by the method described later, furthershifted to the visible spectrum with the maximum located at ˜455 nm.

Mixtures of (compound 2+TOPO) and (compound 2+europium chloride) weretitrated with standard solutions of europium (III) chloride and TOPO,respectively, and fluorescence intensity was determined. Resultssuggested the formation of fluorescent chelate by a stoichiometric ratioof 1:3:3 of metal, ligand and TOPO. However, compounds withsubstitutions on phenyl ring, such as compound 4 or 8 (Figure III),resulted in stoichiometric ratio of 1:2:3. While we do not wish to bebound to a particular mechanism, it appears that certain of thesechelates, in combination with non-fluorescent chelators such as TOPO orNH₃ in aqueous solution, operate as bis chelators. In other words, twofluorescent chelating molecules will complex with a single metal ionwith three non-fluorescent chelators, in a 1:2:3 molar ratio. Moreover,it is possible to avoid hydration with water molecules where one of theAryl moieties is further substituted adjacent to the diketonesubstitutent with an additional chelating moiety. Specifically, FigureIV illustrates this possibility where one of the aryl moieties issubstituted with:O—CX—R

where X=S or O, and R=O—R₁ or S—R₁, where R₁=C₁-C₅ alkyl, C₁-C₅ alkenyl,C₁-C₅ alkynyl groups, or substituted C₁-C₅ alkyl, C₁-C₅ alkenyl, C₁-C₅alkynyl groups. Preferably, the further aryl substituent is —CO—O-alkyl,such as —CO—OCH₃. In Figure IV, M=the metal ion, P=a non-fluorescentchelator, the organic chelating molecular formulas are shown withchelation via the oxygen molecules, and H₂O indicates hydration of thecomplex.

Having obtained the stable chelates with desired properties, wedemonstrated their potential applications in immunoassays as fluorescentprobes using a simple procedure for loading the chelate into carboxylatex as described below. The new procedure was successfully used forloading a variety of carboxy latex of different size (34, 60, 104, 190,200, 300 and 400 nm) and parking areas (7, 50, 90 Å²/carboxyl group,etc), as well as plain polystyrene of different sizes, with chelatesformed by different PNPD derivatives. No significant changes in theoptical properties of the europium chelates were observed on loadinginto carboxy latex particles except that the excitation maximum wasshifted to longer wavelength by 10-20 nm. Biotinylated BSA was passivelyadsorbed onto the chelate (which had already been loaded carboxy latexparticles) followed by binding of NeutrAvidin—(Pierce Biotechnology,Rockford, Ill.) as described below.

A heterogeneous sandwich immunoassay for canine heartworm (HTWM) wasperformed using chelate loaded carboxy latex coated with NeutrAvidin.The critical steps in the assay were as follows: Polystyrene wells werecoated with rabbit anti HTWM AB followed by blocking with BSA to preventnon-specific binding. Serial dilutions of HTWM antigen in serum wereincubated in these wells (the lowest concentration in panel #10 wasabout 0.03 ngr/ml) followed by biotinylated chicken anti HTWM AB. Thewells were then washed and finally incubated with neutrAvidine-coatedfluorescent carboxy latex. After thoroughly washing off the unboundparticles, fluorescence was read in both continuous and time resolvedmode. Results are shown in Figure V.

A heterogeneous sandwich immunoassay was also performed using theseneutrAvidine-coated chelate-loaded carboxy latex for Feline leukemiavirus (FeLV) antigen. The critical steps in the assay were as follows:The polystyrene wells were coated with monoclonal anti FeLV AB followedby blocking with BSA to prevent non-specific binding. Dilutions of anFeLV antigen panel were incubated in the wells, followed by biotinylatedmonoclonal anti FeLV AB. The wells were then washed and finallyincubated with NeutrAvidin coated fluorescent carboxy latex. Afterthoroughly washing off the unbound particles, fluorescence was read inboth continuous and time resolved mode. Results are shown in Figure VI.

Antibody/protein coated colloidal gold is used extensively as a probe inimmunodiagnostics, histochemistry and cyctochemistry²¹. In typicalimmunoassays, colloidal gold provides a label for visual or qualitativedetection²². It was reasoned that the concept of florescent colloidalgold particles would allow the gold particles to be used for bothqualitative (by the visual observation of color) as well as quantitativedetection (by measuring the florescence) of the targets. On these lines,a successful procedure was developed for the first time to coat thecolloidal gold particles with the europium chelates. As described below,Eu chelates of PNPD and its fluorinated analogs were coated ontocolloidal gold particles and the optical properties of the resultingflorescent gold particles were studies as shown in Figure VIII. It wasfurther shown that the florescent gold particles can be successfullycoated with antibodies for using them as probes in immunoassays.

Three additional carboxyl functionalized derivatives of PNPD weresynthesized successfully as shown in Figure VII, to enable the directconjugation of the new ligands to proteins. The synthesis of1-phenyl-3(6-carbomethoxy-naphthyl)-1,3-propanedione (17) wasaccomplished as described in the methods from2,6-dimethyl-naphthlein-dicarboxylate and acetophenone. Thecorresponding carboxylic acid (18) was obtained by the hydrolysis of themethyl ester under sonicating conditions. The carboxylic acidderivatives (20 &21) were also synthesized using similar syntheticprocedures and appropriate starting materials. It is well known that theN-hydroxysuccinimide esters of the carboxylic acid compounds reacts withthe amino groups in proteins resulting in the formation of correspondingconjugates. The NHS ester of1-phenyl-3(6-carboxy-naphthyl)-1,3-propanedione (19) was synthesized andconjugated successfully to BSA and both chicken and rabbit α HTWMAntibodies resulting in the ligand labeled proteins. Absorption spectrumof the labeled proteins have a well defined peak, characteristic of newligands, with maxima at 360 nm suggesting successful conjugation.Labeled proteins formed fluorescent chelates when they were mixed witheuropium chloride and TOPO solutions. These chelate labeled antibodiesallow us to run the sandwich immunoassays without using latex particles.The soluble chelate labeled antibodies shall potentially eliminate theusual diffusion associated problems that are associated with latexparticles. To further enhance the sensitivity of this technology, wealso successfully labeled polymers with multiple amino groups such aspolyallylamine and lysine-aspartic acid polypeptides, as described,below. Further conjugation of these labeled polymers to antibodiesshould result in soluble protein-polymer conjugates with multiplechelate labels. The NHS ester of1-(3,5-F₂-phenyl)-3(6-carboxy-naphthyl)-1,3-propanedione was alsosynthesized by following similar chemistry and was used to successfullyto label both proteins and polymers.

Synthesis of 1-phenyl-3-naphthyl-1,3-propanedione (2): 9.5 gr of 50%slurry of sodium amide was suspended in 100 ml of anhydrous ether in a250 ml round bottom flask that was equipped with a magnetic stir bar anda condenser. To this suspension was added 10.2 gr of solidacetonaphthone and continued stirring for 5 min at room temperature.After 5 minutes, 8.16 gr of methylbenzoate was added in one lot as aneat liquid. At this point, the reaction flask was transferred to an oilbath and the bath temperature was raised so that the contents refluxgently in ether for 3 hours. The contents were then poured onto 300 grof solid ice and the pH was adjusted to 7.0 with conc. HCl whilestirring. The ether layer was separated and the aqueous layer wasextracted with 150 ml aliquots of ether twice. Combined ether layerswere dried over a bed of anhydrous sodium sulfate and concentrated onrotavapor to about 100 ml and let it sit at RT. The recrystallized solidwas filtered, washed with ether and dried to obtain 10.6 gr of soliddiketone.

Synthesis of 1,3-dinaphthyl-1,3-propanedione (3): 4.75 gr of 50% slurryof sodium amide was suspended in 150 ml of anhydrous ether in a 250 mlround bottom flask that was equipped with a magnetic stir bar and acondenser. To this suspension was added 5.1 gr of solid acetonaphthoneand continued stirring for 5 min at room temperature. After 5 minutes,5.58 gr of methyl-2-naphthoate was added in one lot. At this point, thereaction flask was transferred to an oil bath and the bath temperaturewas raised so that the contents reflux gently in ether for 3 hours. Thecontents were then poured onto 300 gr of solid ice. On acidificationwith conc. HCl, solid product fell out of solution. The solid productwas filtered and recrystallized in ether/methanol mixture.

Synthesis of 1-[(3,5-F₂-phenyl]-3-naphthyl-1,3-propanedione (4): 2.135gr of 50% slurry of sodium amide was suspended in 50 ml of anhydrousether in a 100 ml round bottom flask that was equipped with a magneticstir bar and a condenser. To this suspension was added 4.936 gr of solidacetonaphthone and continued stirring for 5 min at room temperature.After 5 minutes, 5 gr of methyl-3,5-difluorobenzoate was added in onelot. At this point, the reaction flask was transferred to an oil bathand the bath temperature was raised so that the contents reflux gentlyin ether for 3 hours. The contents were then poured onto 100 gr of solidice and acidified with cont. HCI while stirring. The ether layer wasseparated and the aqueous layer was extracted with 100 ml aliquots ofether twice. Combined ether layers were dried over a bed of anhydroussodium sulfate and concentrated on rotavapor to obtain a light yellowsolid. The solid was dissolved in minimal volume of ether and filtered.The filtered solution was kept at 4 C to obtain crystalline product. TLCshowed it as a clean single spot without any contamination.

Loading of 3,5-F₂-PNPD-Eu chelate into 104 nm carboxy latex: 9.3 mg of3,5-F₂-PNPD in 30 ml of dioxane, 17.4 15 mg of TOP0 in 45 ml of methanoland 15 ml of 1 mM Eu(II1)chloride were mixed with 100 ml of methanol.After 5 minutes, this mixture was diluted with 100 ml of DI water andput on shaker for 30 minutes to allow complete formation of the chelate.In the mean time, 2 ml of 104 nm CML latex (Seradyne, 10% solids,lot#C98 1838)was mixed with 20 μl of 6N NaOH solution and 2 ml ofmethanol and incubated for 30 minutes. After 30 minutes, the chelatesolution and the particles were mixed together along with 80 μl of 6NNaOH and were put on a shaker for 4 hours at RT. After 4 hours, themixture was concentrated to ˜47 ml on rotavapor at 45 C under vacuum.The concentrated particle suspension was dialyzed against 6 L of 10 mMsodium chloride in DI water over 3 days, changing the dialysis buffertwice a day.

Biotinylation of BSA: 10% BSA solution (3 gr/3O ml of DIW) was filteredthrough 0.2μ filter and dialyzed against 4 L of DI water. One ml of thedialyzed BSA (63.5 mg/ml) was diluted with 7 ml of 50 mM sodiumphosphate buffer pH 8.0 and mixed with 0.5 ml of NHS-X-biotin(PierceChemical Company) solution (20 mg/ml) in anhydrous DMF and let thereaction go for 3 hrs while stirring. After 3 hrs, the reaction mixturewas dialyzed against 6 L of 20 mM TRIS Ph 7.4 to get rid of theunreacted biotin.

Coating of fluorescent particles with biotin-BSA: 3 ml of particles(−0.4% solids) in 10 mM NaCl in DI water were mixed with 60 ˜μl of 1MTRIS Ph 7.4 and 2.5 ml of biotin/BSA (7.15 mg/ml in 20 mM TRIS Ph 7.4)in a polypropylene tube and put on rotator at 4 C overnight. Nextmorning, the particles were spun at 15K rpm for 30 minutes. The pelletwas resuspended in 2 ml of 20 mM TRIS Ph 7.4 and spun again. Thisprocess was repeated twice.

Finally the particles were suspended in 2 ml of 20 mM TRIS pH 7.4.

Coating of biotin-BSA coated particles with NeutrAvidine: 2 ml of abovebiotin-BSA coated particles in 20 mM TRIS pH 7.4 were mixed with 0.4 mlof 10 mg/ml solution of neutrAvidine in the same buffer and put onrotator at 4 C for 48 hrs. After 48 hrs, the unbound protein was removedas in the case of biotin-BSA coating procedure and the particles werefinally suspended in 20 mM TRIS pH 7.4.

Determination of HTWM antigen using neutrAvidine coated fluorescentparticles: The rabbit anti HTWM AB coated wells were incubated with 100ml of HTWM antigen panels #0-10 for 30 minutes. The wells were thenwashed thrice with a commercially available HTWM plate wash (IDEXXLaboratories, Inc., Westbrook, Me.) followed by incubation with 5 pg/mlbiotinylated chicken anti HTWM AB in HTWM conjugate diluent (IDEXXproduct) for 30 min. The wells were washed now 7 times followed byincubation with neutrAvidine coated fluorescent particles for an hour.After an hour, the wells were washed 7 times and read the fluorescencein time resolved as well as continuous mode. Figure V shows the resultsof this assay using both continuous and time resolved fluorescence.

Synthesis of 1-phenyl-3(6-carbomethoxy-naphthyl)-1,3-propanedione: To asuspension of sodium amide (1.56 gr of 50% suspension in toluene) inanhydrous THF, was added neat acetophenone (2.88 gr) and stirred for 3minutes at RT in a 500 ml round bottom flask equipped with a magneticstir bar and a condenser. After 3 minutes, solid2,6-dimethyl-naphthlein-dicarboxylate was suspended into the flask andraised the oil bath temperature to reflux THF. After 4 hrs, the reactionmixture was poured into excess ice cold water to precipitate theproduct. The product was filtered off and recrystallized from ethanol.

Synthesis of 1-phenyl-3(6-carboxy-naphthyl)-1,3-propanedione: One gramof 1-phenyl-3(6-carbomethoxy-naphthyl)-1,3-propanedione was suspended in40 ml of 1N NaOH and sonicated 10 times (30 sec pulses) with a probesonicator. The insoluble solids were filtered off and used for anothercycle of sonication. The clear filtrate was acidified with conc. HCl toprecipitate the carboxylic acid product. The product precipitate wasdried by connecting to lyophilizer overnight. The TLC and mass spectralanalysis conformed the formation of the carboxylic acid.

Synthesis of the NHS ester of1-phenyl-3(6-carboxy-naphthyl)-1,3-propanedione: The1-phenyl-3(6-carboxy-naphthyl)-1,3-propanedione (29.26 mg), dicyclohexylcarbodiimide (59.95 mg) and N-hydroxy-succinimide (31.77 mg) weresuspended together in 1 ml of anhydrous DMF in a brown glass vialequipped with a magnetic stir bar and stirred overnight at RT. Nextmorning, the insoluble precipitate was filtered off and the DMF solutionof the product NHS-ester was stored in a brown glass vial filled withnitrogen and used for protein conjugations without any furtherpurification.

Conjugation of BSA with NHS ester of1-phenyl-3(6-carboxy-naphthyl)-1,3-propanedione: Two ml of 7.82 gr/mlBSA solution in 50 mM SPB pH 8.0 was mixed with 2 aliquots of 3 μl ofthe above stock NHS ester solution in a glass vial equipped with a smallmagnetic pellet and stirred at RT for 75 min. After 75 min, the reactionmixture was passed through a size exclusion column equilibrated with 50mM borate buffer pH 9.0 and the protein fraction was collected byfollowing the absorption at 280 nm.

Conjugation of chicken α HTWM AB and rabbit α HTWM AB with NHS ester of1-phenyl-3(6-carboxy-naphthyl)-1,3-propanedione: One ml of 6 mg/mlantibody solution in 50 mM SPB pH 8.0 was mixed with 6 μl of NHS esterstock solution in DMF solution in a glass vial equipped with a smallmagnetic pellet and stirred at RT for 75 min. After 75 min, the reactionmixture was passed through a size exclusion column equilibrated with 50mM borate buffer pH 9.0 and the protein fraction was collected byfollowing the absorption at 280 nm.

Conjugation of polyallylamine with NHS ester of1-phenyl-3(6-carboxy-naphthyl)-1,3-propanedione: Two ml of 6 mg/mlsolution of polyallylamine in 50 mM SPB pH 8.0 is treated with 20 μl ofstock NHS-ester solution in DMF in a brown glass vial equipped with asmall magnetic pellet and stirred at RT for 75 min. After 75 min, thereaction mixture was passed through size exclusion column and separatedthe unconjugated ligand.

Eu-PNPD Chelate coated Colloidal Gold: 10 ml of colloidal gold solution(BB International Gold Colloidal; 40 nm; CAT #EM. GC 40; Batch #2862)was spun at 800 rpm for 15 min and the pellet was retained. The goldpellet was then dissolved in 20 ml of 0.1 M borate buffer pH 9.0 in a 50ml glass conical flask. To this colloidal gold solution were added 400ml each of 10% Tween 20 in DI water followed by 1 mM solution of PNPD.The mixture was the put on a shaker for 10 min. After 10 min, 400 mleach of 1 mM solutions of TOPO followed by europium chloride were added.Finally the mixture was put on shaker overnight. Next day the solutionwas spun at 8000 rpm for 20 min and the pellet was retained. The pelletwas redissolved in 20 ml of borate buffer and spun again at 8K rpm for20 min. The pellet was finally dissolved in 2 ml of borate buffer toobtain a solution of fluorescent colloidal gold.

Eu-3,5-F2-PNPD chelate coated Colloidal Gold: The procedure is same asin the case of Eu-PNPD chelate except that the borate buffer wasreplaced with DI water.

Binding of rabbit α HTWM antibody to chelate coated colloidal gold: 119μl of 4.5 mg/ml rabbit α HTWM solution was mixed with 881 μl of chelatecoated colloidal gold solution and incubated at room temperature for 15minutes. After 15 min, the contents were spun at 8000 rpm for 12minutes. The pellet was dissolved in 1 ml of 0.1 M borate pH 9.0 andspun again at 8000 rpm for another 12 min. The gold pellet was finallydissolved in 1 ml of 0.1 M borate pH 9.0. The antibody present insupernatant was measured and confirmed the binding of the protein tochelate coated gold. A number of embodiments of the invention have beendescribed. Nevertheless, it will be understood that variousmodifications may be made without departing from the spirit and scope ofthe invention. Further conjugation of the chelating system can beachieved. In case of anionic form (2a) of Compound 2, the conjugation isextended further by three additional double bonds of the phenyl ring,and in the case of the anionic form of compound 3a, conjugation isextended by five double bonds of the naphthyl ring. For example, anumber of other potential chelating compounds may be evaluated accordingto the invention. In particular, as noted, compounds which exhibitabsorption maxima in the visible or near visible spectrum are preferred.Aqueous solubility is also preferred. Substituent selection to producepKa values below 9.0 are preferred, particularly when using latexparticles.

Other transition metals can be used, including not only lathanide seriesmetals but also Tr, Se, and Ru, particularly the latter.

Other immunoassays can also be performed using the fluorescing compoundsof the present invention. These include, but are not limited to, assaysfor HIV, FIV, hepatitis, ehrlichiosis, borrelia burgdorferi (Lymedisease), parvovirus, leishmania, hCG, insulin, c-peptide and T4.

Accordingly, other embodiments are within the scope of the followingclaims.

REFERENCES

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1-32. (canceled)
 33. Apparatus for detecting fluorescence from a samplecomprising an irradiating energy source that produces irradiating energyλ equal to or greater than 360 nm; a detector positioned to detectfluorescence from the sample; and a sample holder for holding the samplein position to be irradiated by the energy source.
 34. The apparatus ofclaim 33 in which the irradiating energy source is a semiconductor lightsource.
 35. The apparatus of claim 34 in which the irradiating energysource is a light-emitting diode.
 36. The apparatus of claim 34 in whichthe irradiating energy source is a laser diode.
 37. The apparatus ofclaim 34 in which the detector comprises circuitry to allowinstantaneous or time-resolved detection of the fluorescence.
 38. Theapparatus of claim 37 in which the detector comprises circuitry to allowtime-resolved detection of the fluorescence.
 39. The apparatus of claim34 in which the sample holder is sized and designed for a detectionprocess selected from the group consisting of: cell imaging, flowcytometry, immunohistochemistry, and immunoassay.
 40. The apparatus ofclaim 34 further comprising an aqueous sample position in said sampleholder.
 41. The apparatus of claim 40 in which said aqueous samplecomprises a fluorescent Europium chelate in aqueous solvent.